Isolation Of Trophoblast Cells Research Articles

Microfluidic organic bioelectronic chips for efficient isolation of trophoblast cells using a combination of rational catenation and electrically controllable refining

The non-invasive prenatal test (NIPT) has become the global standard for effective prenatal screening for newborn health care. Recently, there has been rapid growth in next-generation sequencing (NGS) technologies based on genomics using fetal nucleated red blood cells (fnRBCs) purified on chips; the performance of this approach can be competitive with cell-free fetal DNA (cffDNA) detection, but avoids the inherent drawbacks of cffDNA in NIPTs. To isolate the targeted cells from clinical samples, microfluidic device systems can be designed to minimize the use of reagents/samples and enhance the capture of targeted specimens by increasing the degree of contact between the targeted cells and chips. Nevertheless, sample refining remains a critical challenge affecting device performance. In this study, we developed poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) nanofiber–based film capacitors as organic bioelectronics interfaces (OBEIs). Efficient cell capture and release was possible through cyclic voltammetry (CV)–based charging/discharging operations, which varied the surface charge of the OBEIs. The combination of a microfluidic channel and film capacitor was engineered to refine 0.2% spike-in JEG-3 cells (a trophoblast cell line derived from the male) from 1 × 106 HeLa cells (a cervical cancer cell line derived from the female) into JEG-3 cells of 93.8% purity. This device platform displayed outstanding efficiency in the isolation of targeted cells, with a 1000-fold enrichment through three capture/release cycles of programmable CV operations, thereby providing high-purity targeted JEG-3 cells for use in a downstream real-time quantitative PCR (qPCR) assay. The qPCR and fluorescence in situ hybridization (FISH) data revealed that the off-chip-collected liquid biopsy sample could be used to identify the XY chromosome from the refined JEG-3 cells. This electrically controllable microfluidic OBEI platform appears to be a potential tool for enriching targeted cells from liquid biopsy samples and enhancing precision medicine applications based on cell-based NIPT technologies.

Methods of detection and isolation of trophoblast cells from trans-cervical specimens – a historical overview

Abstract Trophoblast cells can be detected and isolated from the cervical epithelial cells obtained via various techniques of trans-cervical samples collection such as a mucus aspiration, endocervical lavage, or standard cervical brushing in the early first trimester, even from the 5 weeks’ gestation. Isolated fetal cells can be used in the early prediction of fetal sex, prenatal diagnostics of the most common aneuploidies, and any other genetic abnormalities. Nevertheless, the collection of trophoblastic cells has limited efficacy compared to currently used methods of detection of free fetal DNA in maternal circulation or other protocols of invasive prenatal diagnostics available at later stages of pregnancy. In the past years, trans-cervical cell samples were collected mainly in women before planned pregnancy termination. The early trophoblastic cells isolation from women in ongoing pregnancies opens new perspectives for further studies focused on the elucidation of pathophysiology of numerous pregnancy-related complications.

Использование трофобластов цервикального канала для неинвазивной пренатальной детекции частых анеуплоидий

The article presents an experience of isolation of trophoblast cells from cervical samples of pregnant women and demonstrates a possibility of determining aneuploid cells in cervical samples using FISH analysis. Objective. To study methods of effective isolation and reliable detection of trophoblasts for genetic pathology analysis. Patients and methods. The participants of the study were three pregnant women, who according to the findings of the combined first trimester screening test were referred to the group with a high risk for fetal aneuploidy. The terms of gestation varied from 12 to 14 wks. Immunocytochemical detection of trophoblast cells was performed with FITC-labelled HLA-G antibodies. FISH was performed with the use of locus-specific DNA probes on chromosomes 13/21 and a DNA probe on chromosome 18 centromere. The chromosome set of a fetus was verified by the results of standard cytogenetic analysis of semi-direct chromosome preparations from cytotrophoblast cells obtained by chorionic villi biopsy. Results. HLA-G-positive cells were found in all examined cytological specimens. FISH analysis with the use of DNA probes on chronomomes, whose trisomies are compatible with live birth, detected aneuploid cells in all three cases. Conclusion. The use of cervical trophoblasts for noninvasive collection of information about the genetic status of the developing fetus at present needs further study of effective and reliable methods of their isolation, detection and analysis. Key words: aneuploidy, throphoblast cells, NIPT, prenatal diagnosis, HLA-G

Fetal genome profiling at 5 weeks of gestation after noninvasive isolation of trophoblast cells from the endocervical canal.

Single-gene mutations account for more than 6000 diseases, 10% of all pediatric hospital admissions, and 20% of infant deaths. Down syndrome and other aneuploidies occur in more than 0.2% of births worldwide and are on the rise because of advanced reproductive age. Birth defects of genetic origin can be diagnosed in utero after invasive extraction of fetal tissues. Noninvasive testing with circulating cell-free fetal DNA is limited by a low fetal DNA fraction. Both modalities are unavailable until the end of the first trimester. We have isolated intact trophoblast cells from Papanicolaou smears collected noninvasively at 5 to 19 weeks of gestation for next-generation sequencing of fetal DNA. Consecutive matched maternal, placental, and fetal samples (n = 20) were profiled by multiplex targeted DNA sequencing of 59 short tandem repeat and 94 single-nucleotide variant sites across all 24 chromosomes. The data revealed fetal DNA fractions of 85 to 99.9%, with 100% correct fetal haplotyping. This noninvasive platform has the potential to provide comprehensive fetal genomic profiling as early as 5 weeks of gestation.

Isolation of trophoblast cells from transcervical specimens for prenatal determination of fetal sex by single-cell analysis

Trophoblast cells migrate from the placenta into the cervical canal early in the first trimester and can be collected in transcervical specimens (TCS). DNA isolated from these fetal cells could potentially be used for prenatal genetic diagnosis. The objectives were to 1) separate fetal cells from resident maternal cells in TCS and 2) conduct gender determination by single cell PCR.

Antibodies to Trophoblast Antigens HLA-G, Placenta Growth Factor, and NeuroD2 Do Not Improve Detection of Circulating Trophoblast Cells in Maternal Blood

Objectives: Non-invasive prenatal diagnosis using circulating fetal trophoblast cells has been challenging due to lack of a reproducible trophoblast-specific antibody. We investigated the use of three trophoblast cell-specific antibodies, HLA-G, placenta growth factor, and neuroD2, for the isolation of trophoblast cells from the maternal circulation. Methods: Trophoblast cells were isolated by density centrifugation from maternal blood samples (gestational age 10–20 weeks, n = 9). All women were carrying a male fetus. Following immunocytochemical staining with the trophoblast-specific antibodies, fluorescent in situ hybridization was performed, to verify whether any stained cells were indeed fetal. Results: The HLA-G antibody had a ubiquitous staining pattern, which was not specific for trophoblast cells. Neither the placenta growth factor nor the neuroD2 antibodies were able to identify any trophoblast cells. Following fluorescent in situ hybridization, no male cells were detected on any of the slides. Conclusion: The antibodies used in this study were unable to improve detection of trophoblast cells in the maternal circulation.

OXIDATIVE STRESS REPRODUCES PLACENTAL ABNORMALITIES OF PREECLAMPSIA

Objective: The activities of placental superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), but not catalase, are lower than normal in preeclampsia, which could contribute to the uncontrolled placental production of lipid peroxides and thromboxane (TX). Oxidative stress, hyperlipidemia and increased iron levels in the maternal compartment in preeclampsia could be responsible for these placental changes by causing oxidative stress in the placenta. Methods: We tested this possibility in vitro by exposing a trophoblast-like cell line, ED27, to a combination of linoleic acid (LA, 90 μM) and an oxidizing solution composed of hypoxanthine, xanthine oxidase and ferrous sulfate (OxLA) for 6 days. For these studies, the cells were treated with dexamethasone (10−8 M) for the first 72 hr. This was done to differentiate the cells into a phenotype more like syncytiotrophoblast cells as evidenced by production of β-human chorionic gonadotropin (β-hCG). Results: After 6 days of exposure to OxLA, the activities of SOD and GSH-Px were significantly decreased as compared to exposure to LA alone. In contrast, catalase activity was increased by OxLA. The OxLA-induced decreases in SOD and GSH-Px activities were attenuated by deferoxamine, an iron chelator, suggesting a role for Fe2+ in the decreased activities. Compared to LA, OxLA significantly increased TX secretion and lipid peroxidation in cells and media at 2, 4 and 6 days. Deferoxamine inhibited the OxLA-induced increase in lipid peroxidation, but not the increase in TX. Isolation of trophoblast cells and villous core tissue from term placentas verified that antioxidant enzyme activity was localized primarily to the trophoblast cell compartment lending validity to the in vitro findings. Conclusions: These data mimic the changes in placental SOD, GSH-Px, catalase, TX and lipid peroxidation that occur in preeclampsia suggesting that maternal hyperlipidemia and increased iron levels may be responsible for placental oxidative stress and abnormalities in antioxidants and thromboxane.

Efficient isolation of trophoblast cells from peripheral blood.

Efficient isolation of trophoblast cells from peripheral blood.

Isolating Fetal Trophoblast Cells for Prenatal Genetic Diagnosis

To the Editor. —The article by Drs Simpson and Elias 1 reviews the feasibility of using various candidate fetal cells (eg, trophoblast cells, lymphocytes, granulocytes, and erythroblasts) for prenatal genetic diagnosis. The authors consider isolation of trophoblast cells to be theoretically attractive, but stated that with regard to the use of monoclonal antibodies directed toward trophoblast, no further data have been presented since the original publication in 1990. 2 In our laboratory, monoclonal antibodies to trophoblast have been used in an immunomagnetic separation to isolate trophoblasts from maternal blood. Polymerase chain reaction analysis of the cell isolates correctly identified fetal sex in 13 of 14 cases. 2 The cell isolates were identified as syncytiotrophoblast in a morphological study. 3 More recently, further evidence was presented using polymerase chain reaction analysis to detect the paternally inherited allele for β-thalassemia in trophoblast isolates from maternal blood samples. 4 Inheritance was confirmed by

Enzymatic isolation of human trophoblast and culture on various substrates: Comparison of first trimester with term trophoblast

A simple method is described for the isolation of trophoblast cells from both first trimester and term placenta. Trophoblast preparations were characterized by light microscopy, scanning and transmission election miscroscopy and immunohistochemistry to distinguish these cells from mesenchyme and endothelium. Trophoblast cells were cultured on various substrates and a comparison made of their ability to attach, proliferate and function. A collagen gel substrate produced by repolymerization of an acid soluble collagen fraction from chorionic villi allowed rapid attachment of trophoblast cells and maintainance of their original morphology. Term trophoblast cells were shown to become fully functional in short term (three day) cultures by virtue of their increased immunocytochemical staining for the presence of beta hCG, hPL and SPI. beta hCG increased significantly by day three thus demonstrating functional activation. Trophoblast cells from first trimester placenta formed proliferating colonies of hormone producing cells while those from term placenta reaggregated into clusters and closely resembled syncytiotrophoblast both morphologically and functionally. This short term culture system for term trophoblast will allow further studies into the biology of trophoblast polypeptide hormone synthesis and secretion.